POS0147 αVβ3 INTEGRIN AS A LINKER BETWEEN FIBROSIS AND THYROID HORMONES CROSSTALK
Authors: S. Tartakover Matalon, M. Kohon, M. Zaaroor, T. Hornik-Lurie, A. Shalom, A. Berl, L. Drucker, Y. Levy
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Systemic sclerosis (SSc) is an autoimmune disease characterized by tissue fibrosis of the skin and internal organs. Key players in SSc are myofibroblast cells (MF) that produce large amounts of extracellular matrix (ECM), which facilitates fibrosis. TGFβ is a key factor involved in MF differentiation and collagen-rich ECM (fibrotic-ECM) secretion. Most of the TGFβ is bound to the ECM through latency-associated peptide (LAP) that contains the amino acid sequence arginine-glycine-asparagine (RGD motif). In order to be active, TGFβ must be released from the ECM by proteases like MMP9 or αv integrins that bind LAP through their RGD recognition site. MF express high levels of the integrin αvβ3. Interestingly, αvβ3 is a special integrin since it also serves as a membrane thyroid hormone (TH) receptor that binds T3 and especially T4. The TH binding sites are located proximal to the RGD binding site. MF also express high levels of the pro-fibrotic miRNA-21, which regulates the TGFβ pathway and the level of DIO3 enzyme, which degrades T3. T3 attenuates fibrotic processes. Since high levels of αvβ3 integrin are expressed by MF in the fibrotic sites and the TH binding site of αvβ3 is close to the RGD recognition site, we hypothesized that the TH binding site of αvβ3 is involved in mediating the fibrotic process. <h3>Objectives</h3> 1. To examine the effect of fibrotic-ECM produced by dermal fibroblasts (DF) triggered by TGFβ, on naive DF phenotype and their αvβ3 expression; 2. To examine the connection between the thyroid hormone binding site of αvβ3 integrin to fibrosis-related processes. <h3>Methods</h3> DF were isolated from healthy skin, cultured without/with TGFβ, and removed by NH<sub>4</sub>OH, leaving the normal/ fibrotic ECMs in the wells. ECMs were evaluated for collagen-I and TGFβ levels (Immunohistochemistry, western blot). The effect of the normal/fibrotic ECMs on the following parameters of naïve DF was evaluated: phenotype; expression of MF biomarkers (collagen, elastin and αSMA), expression of αvβ3 integrin, miRNA-21, and DIO3 enzyme; activation of the TGFβ pathway (Smad3 phosphorylation); and MMP9 activity (automatic cell counter, western-blot, RT-qPCR, gelatin zymography). Moreover, the effect of tetraiodothyroacetic acid (tetrac, T4 derivative, and αvβ3 ligand) on the fibrotic-ECM ability to induce DF differentiation into MF and to express αvβ3, miRNA-21 and DIO3 was studied. Last, free T3 levels were measured in 18 SSc patients of whom 7 had pulmonary arterial hypertension (PAH). The patients were divided according to high (n=6)/low (n=12) T3 levels into 2 groups and the difference in circulating miRNA-21 levels between the groups was evaluated. <h3>Results</h3> Fibrotic-ECMs generated by TGFβ-triggered DF had higher levels of collagen-I and TGFβ in comparison to normal ECM. In comparison to the normal ECM, the fibrotic-ECM increased the followings in the DF: 1) ability to liberate TGFβ from the ECM by increasing MMP-9 activity and αv expression. 2) TGFβ activation and differentiation to MF evidenced by increased expression of phosphorylated Smad3, αSMA, collagen, and elastin, and increased proliferation. 3) Expression of αvβ3, miRNA-21 and DIO3 (all changes in 1-3: 24-123%↑, p≤0.05). The addition of tetrac to DF cultured on fibrotic-ECM downregulated cell proliferation, collagen, elastin and aSMA expression to the levels found in DF cultured on normal ECM. Furthermore, it reduced αvβ3, miRNA-21, and DIO3 levels. Accordingly, decreased miRNA-21 levels were found in the high T3 SSc group, compared to the low T3 group that contained all the PAH patients (p≤0.05). <h3>Conclusion</h3> Our results demonstrated the existence of a vicious cycle: TGF-triggered DF produced a fibrotic-ECM that promoted bystanders' DF differentiation to MF. Using this model, we demonstrated that tetrac binding to αvβ3 integrin inhibited the differentiation of DF to MF, and their ability to activate the αvβ3/miRNA-21/DIO3/T3 pathway. These results suggest that the thyroid hormone binding site of αvβ3 may be a potential target for the treatment of fibrosis. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.